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Phenotypic analysis of hematopoietic stem and progenitor cells has been an invaluable tool in defining the biology of stem cell populations. We use here flow cytometry to examine the expression of human erythroid-specific surface markers during the maturation of early committed erythroid cells derived from cord blood in vitro. The temporal order of the expression of erythroid specific markers was as follows: Kell glycoprotein (gp), Rh gp, Landsteiner Wiener (LW) gp, glycophorin A (GPA), Band 3, Lutheran (Lu) gp, and Duffy (Fy) gp. The time at which some of these markers appeared suggests possible roles for some of these erythroid-specific polypeptides during the differentiation of these committed progenitors. The early appearance of Kell gp raises the possibility that it may have an important role in the early stages of hematopoiesis or cell lineage determination. Kell gp may also be a useful marker for the diagnosis of erythroleukemia. The late expression of Lu gp suggests it may be involved in the migration of erythroid precursors from the marrow. Fy gp is also expressed late consistent with a role as a scavenger receptor for cytokines in the bone marrow and circulation. Rh c antigen appeared before Rh D antigen, and it is suggested that this may reflect a reorganization of the developing erythroid cell membrane involving the Rh polypeptides and other components, including GPA and Band 3.

We have previously shown that a murine monoclonal antibody (A3D8) identifies a human erythrocyte protein antigen whose expression is regulated by the Lutheran inhibitor [In(Lu)] gene. In the present study, we demonstrated by immunoprecipitation and Western blot techniques that the antigen defined by A3D8 was on an 80-kD erythrocyte membrane protein. A second 170-kD protein was coprecipitated with the 80-kD protein but failed to show antigen activity by Western blot analysis. The 170-kD protein, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in two dimensions, was composed of 50- and 30-kD disulfide-linked subunits. In(Lu) Lu[a-b-) erythrocytes differed from Lu(a+b+) or Lu(a-b+) erythrocytes in that In(Lu) deoxycholate erythrocyte membrane extracts contained trace amounts of immunoprecipitable 80-kD protein compared with detergent-solubilized erythrocyte membrane extracts prepared from Lu(a+b+) or Lu(a-b+) subjects.

The In(Lu) phenotype is inherited as an autosomal dominant trait and is characterized by suppression of the Lutheran, P1, i, and Aua erythrocyte blood group antigens. We have developed a monoclonal antibody (L21) that strongly agglutinates all erythrocytes except In(Lu), and we have identified eight In(Lu) individuals among 42,000 blood donors tested. Studies of two families confirmed the dominant mode of inheritance and revealed several new features of this phenotype. The erythrocytes of all five affected individuals from the two families exhibited diminished hemagglutination by the lectin concanavalin A, although they reacted normally with several other lectins. The erythrocytes of two affected individuals in one family exhibited marked acanthocytosis. The erythrocytes of the proposita of the other family exhibited a mild degree of poikilocytosis, but the cells of the other two affected individuals in this family had normal morphology. The osmotic fragility of fresh In(Lu) erythrocytes was normal, but after incubation for 24 hours at 37 degrees C in plasma the In(Lu) cells exhibited a marked increase in resistance to osmotic lysis. During the incubation period the erythrocytes lost K+ and their total cation content was diminished. These data indicate that in addition to the suppression of blood group antigens noted previously, the In(Lu) phenotype includes a variety of morphological abnormalities and a defect in electrolyte metabolism. The use of L21 and similar monoclonal antibodies provides a more sensitive means of detecting In(Lu) erythrocytes than typing with human anti-Lub antisera.